Comparison of the inoculum effect of in vitro antibacterial activity of Imipenem/relebactam and Ceftazidime/avibactam against ESBL-, KPC- and AmpC-producing Escherichia coli and Klebsiella pneumoniae

Objective To evaluate effect of inoculum size of extended-spectrum β-Lactamase (ESBL)-producing-, AmpC-producing-, and KPC-producing Escherichia coli and Klebsiella pneumoniae on the in vitro antibacterial effects of imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA). Methods We compared the impact of inoculum size on IMR and CZA of sixteen clinical isolates and three standard isolates through antimicrobial susceptibility tests, time-kill assays and in vitro PK/PD studies. Results When inoculum size increased from 105 to 107 CFU/mL, an inoculum effect was observed for 26.3% (5/19) and 52.6% (10/19) of IMR and CZA, respectively; time-kill assays revealed that the concentration of CZA increased from ≥ 4 × MIC to 16 × MIC to reach 99.9% killing rate against K. pneumoniae ATCC-BAA 1705 (KPC-2-, OXA-9- and SHV-182-producing) and 60,700 (SHV-27- and DHA-1-producing). While for IMR, a concentration from 1 × MIC to 4 × MIC killed 99.9% of the four strains. When the inoculum size increased to 109 CFU/mL, neither IMR nor CZA showed a detectable antibacterial effect, even at a high concentration. An in vitro PK/PD study revealed a clear bactericidal effect when IMR administered as 1.25 g q6h when inoculum size increased. Conclusion An inoculum effect on CZA was observed more frequent than that on IMR. Among the β-lactamase-producing strains, the inoculum effect was most common for SHV-producing and KPC-producing strains. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-023-00660-5.


Background
Production of β-lactamases is the main reason underlying the antimicrobial resistance (AMR) of Gram-negative bacteria; in addition, the selection of β-lactams is closely related to the frequency and evolution of AMR [1].Enterobacteriaceae producing extended-spectrumβ-lactamases (ESBL) and carbapenemases are the main source of multi-drug resistant, and even pan-drug resistant, bacteria worldwide.While the number of treatments is limited, making effective treatment a clinical challenge.
However, the clinical efficacy may be poor even though the strains are sensitive in an in vitro antimicrobial susceptibility test.Differences in the bacterial load have an impact on the choice of drugs and the dosing regimens; in particular, some antibiotics are susceptible to an inoculum effect (defined as attenuated antibacterial activity with increased inoculum size), which has an impact on treatment of infections.The recommended standard bacterial inoculum for the microbroth method is 1-5 × 10 5 CFU/mL [3]; however, the bacterial load varies greatly according to the type and location of an infection.With respect to urinary tract infections (UTIs), the bacterial load is usually low, at about 10 5 -10 6 CFU/mL, while in intra-abdominal infections (IAIs) the bacterial load can be as high as 10 8 -10 9 CFU/mL, and that in meningitis can reach 10 9 CFU/mL [4,5].A series of studies have examined the inoculum effect, and found that carbapenems are less affected than classical BLBLIs (such as piperacillin/tazobactam) and cephalosporins [6,7].In clinical practice, carbapenems are recommended for the treatment of severe infections.Studies show that there is a difference between the clinical cure rates of CZA when used to treat complicated urinary tract infections (cUTIs) and complicated intra-abdominal infections (cIAIs) (92% vs. 80%, respectively) [8]; the cure rates of IMR are similar (97.1% vs. 96.3%,respectively) [9,10].These findings may be related to the inoculum effect.However, there is no consensus about the impact of the inoculum effect on treatment efficacy.
The purpose of this study was to use antimicrobial susceptibility tests, time-kill assays, and in vitro PK/PD studies to evaluate the in vitro inoculum effect of ESBL-, KPC-, and AmpC-producing E. coli and K. pneumoniae on IMR and CZA, thereby providing a reference for clinical application.

In vitro PK/PD studies PK parameters used
The simulated human serum concentrations of CZA and IMR obtained after multiple intravenous administrations were based on PK data from previous studies (Additional file 1: Table S1) [11,12].In the present study, a onecompartment PK model of the agents was used for all experiments.

In vitro PK/PD simulation model and measurement of antibacterial activity
The study was conducted using the in vitro PK Auto Simulation System 400 (PASS-400; Dainippon Seiki, Kyoto, Japan).The bacterial suspension was injected into 100 mL of broth medium to achieve a initial inoculum of 10 5 , 10 7 or 10 9 CFU/mL.At predetermined time points (0, 2, 4, 6, 8, 10, 14, 18, and 24 h), 1.5 mL of the test strain was collected and clones were counted after 18-24 h incubation.Plated using an automatic spiral spreading instrument for colony counts.The limit of clone detection was 30 CFU/mL.Each experiment was performed in triplicate to assure reproducibility.The PD parameters, including Maximum Kill Down (MKD; the difference between the minimum bacterial count and the initial count during the experiment), the difference in bacterial counts between 0 and 24 h (∆log N24), and the bacterial growth recovery time (RT; the time from first exposure to the antibiotic until the moment when the bacterial count again reached its initial level) were analysed by PASS 400 Analyse Bactericidal Activity software.The area between the control growth curve and bactericidal curves (IE) was calculated by the trapezoidal rule using GraphPad Prism 9; these data were used as the integral parameters for evaluating antimicrobial effects.Data were analysed using one-way analysis of variance, and P < 0.05 was considered statistically significant.

Discussion
In recent years, the use of carbapenems to treat severe infections has been increasing worldwide.Under the pressure of antimicrobial selection, the prevalence of carbapenem-resistant bacteria has been increasing year-on-year.Polymyxin, tigecycline and other antibiotics commonly used to treat multidrug-resistant bacterial infections show systemic toxicity and have uncertain efficacy [13]; therefore, new antimicrobial drugs are needed urgently to treat carbapenemresistant and multidrug-resistant bacterial infections.Classical BLIs such as tazobactam, clavulanic acid and sulbactam show insufficient inhibitory activity against AmpC-or KPC-producing strains.Avibactam, Fig. 2 In vitro dynamic time-kill assays for ATCC-BAA 1705 (A-C), E. coli 56706 (D-F), K. pneumoniae 61089 (G-I) and K. pneumoniae 60700 (J-L).Data are expressed as the mean ± SD.The left-hand panels depict an inoculum size of 10 5 CFU/mL; the middle panels show an inoculum size of 10 7 CFU/mL, and the right-hand panels denote an inoculum size of 10 9 CFU/mL a novel diazabicyclooctanone compound, exhibits potent inhibitory activity against AmpC-, OXA-48and KPC-producing strains [14], and relebactam also exhibits good activity against SBL-, AmpC-and KPCproducing strains [15].CZA and IMR show potent antibacterial activity against carbapenem-resistant Enterobacteriaceae [16,17].
The mechanisms underlying the inoculum effect are quite complicated.As the inoculum size increases, the concentration of antibacterial drugs that interact with individual bacterial cells decreases [18], weakening the antibacterial effect of the drugs.Strains with a high inoculum size can reach stationary phase faster, and expression of PBPs during the stationary phase decreases;  A-C), E. coli 56706 (D-F), K. pneumoniae 61089 (G-I), and K. pneumoniae 60700 (J-L).The left-hand panels depict an inoculum size of 10 5 CFU/mL; the middle panels show an inoculum size of 10 7 CFU/mL, and the right-hand panels denote an inoculum size of 10 9 CFU/mL this weakens the effect of drugs targeting PBPs [19].At the same time, when the bacterial inoculum size is high, bacterial quorum-sensing can mediate expression of proteins that reduce antimicrobial susceptibility, such as β-lactamases [19].A previous study found that piperacillin/tazobactam induced a large amount of β-lactamase when the bacterial inoculum size was high [20].In the present study, as the concentration of BLI AVI and REL increased from 4 mg/L to 8 mg/L, the inoculum effect on CZA decreased from 66.7% (8/12) to 33.3% (4/12), and that on IMR decreased from 25% (3/12) to 8.3% (1/12).As the inoculum size increased, it was necessary to increase the concentration of BLI to retain the antibacterial activity of CAZ and IPM.When the inoculum size was 10 9 CFU/mL, the MIC values of CZA and IMR were > 512/4 mg/L.In the time-kill assays, even high concentrations of antibiotics (32 × MIC) did not kill the bacteria.Data from the in vitro PK/PD studies showed that the conventional recommended doses of CZA 2.5 g q8h and IMR1.25 g q6h allowed bacterial regrowth after 2-14 h.At a high inoculum size, a large amount of β-lactamase was produced, negating the effects of AVI and REL.CAZ and IPM are hydrolysed by β-lactamases, which reduces their antibacterial effects.Non-β-lactamase-producing E. coli ATCC 25922 showed an inoculum effect when the inoculum size increased to 10 9 CFU/mL, which may suggest that β-lactamaseproduction is not the only factor involved.
The inoculum effect may also be affected by the type of β-lactamases in β-lactamase-producing strains.In this study, the inoculum effect was greatest against KPCproducing strains and SHV-producing strains.Queenan et al. [21] found that the inoculum effect correlates with the catalytic efficiency of β-lactamase, on the other hand Ehmann et al. [14] stated that the catalytic rate k2/ki of AVI for KPC-2 is 1.3 ± 0.1 × 10 4 M −1 s −1 .Therefore, the high catalytic efficiency of KPC may be the reason underlying the inoculum effect of KPC-producing strains.A previous study on the inoculum effect of ESBL-producing E. coli on piperacillin/tazobactam found no difference in frequency with respect to TEM-producing, SHV-producing, and CTX-M-producing strains [20].The present study included K. pneumoniae but not E. coli; therefore, the type of bacteria may have an impact on the presence of an inoculum effect.
An inoculum effect on cephalosporins was observed more frequent than that on carbapenems [18,19].A previous study showed that the frequencies of inoculum effect on CAZ, cefepime and cefotaxime were observed for 35%, 85% and 100% of ESBL-producing E.coli, respectively, while meropenem did not show an inoculum effect [22].Another study found that the inoculum effect might attributable to a decrease in expression of penicillin-binding protein (PBP) [23].CAZ has a higher affinity for PBP3 and IPM mainly binds to PBP2 [24].When the bacterial inoculum size increases, accumulated signalling molecules such as auto-inducers 2 (AI-2) and Acyl-homoserine lactones (AHLs) mediate quorumsensing [25].Then upregulation of β-lactamases expression and downregulation of efflux pump expression and outer membrane protein would led to the reduction of antibiotics susceptibility [25].The difference in the target protein between CAZ and IPM may be a possible explanation of our finding that an inoculum effect on CZA was observed more frequent than that on IMR.
Many studies have showed that the inoculum effect can impact clinical outcomes [26,27].One study found that when the inoculum of Pseudomonas aeruginosa increased from 5 × 10 4 CFU/mL to 5 × 10 5 and 5 × 10 6 CFU/mL, the MIC of IMR remained almost unchanged [28].Here, we found that the frequency of inoculum effect on IMR was relatively low (25%).The clinical efficacy rates of IMR for the treatment of cUTIs and cIAIs are 97.1% and 96.3%, respectively [9,10], with the difference being non-significant.By contrast, the clinical efficacy rates of CZA for cUTIs and cIAIs are 92% and 80%, respectively [8].The clinical efficacy of CZA for treating infections at different sites varies greatly, which may be related to the presence of an inoculum effect.We found that the inoculum effect on CZA was 66.7%.However, a previous study suggests that the impact of inoculum size on the in vitro antibacterial activity of CZA is less than that of IMR [29].This discordance may be due to use of MICs below or above the measurement threshold, making it difficult to analysis MICs statistically.Also, the previous study examined carbapenem-resistant Enterobacteriaceae, whereas we tested β-lactamase-producing E. coli and K. pneumoniae.
Our study has some limitations.First, the experimental strains produced a variety of β-lactamases simultaneously; the actions of these β-lactamases may have affected the antibacterial efficacy of the drugs.Second, we used only conventional recommended regimens (IMR 1.25 g q6h and CZA 2.5 g q8h) and low-dose regimens (IMR 625 mg q6h and CZA 1.25 g q8h) in the in vitro PK/ PD study.The efficacy of other regimens (such as highdose and continuous dosing regimens) on severe infections needs further study.

Conclusion
IMR and CZA are considered reasonable options for the treatment of multidrug-resistant bacterial infections; however, the presence of an inoculum effect may lead to their failure to treat infections with a high bacterial load (e.g., endocarditis, osteomyelitis, and meningitis); in such cases, IMR may be a better choice.In addition, the presence/absence of an inoculum effect  • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

•
At BMC, research is always in progress.

Learn more biomedcentral.com/submissions
Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? Choose BMC and benefit from:

•
fast, convenient online submission

•
thorough peer review by experienced researchers in your field • rapid publication on acceptance

Table 1 β
-lactamase genotypes, and the effects of different inoculum sizes on the antibacterial MICs of CZA and IMR against E. coli and K. pneumoniae